Biomedical subjects

T O Daniel

Publications and source records attributed to T O Daniel.

At least 19 recordsLinked to original sources

PubMed2001

A novel TNF receptor family member binds TWEAK and is implicated in angiogenesis.

TWEAK is a member of the TNF ligand family that induces angiogenesis in vivo. We report cloning of a receptor for TWEAK (TweakR) from a human umbilical vein endothelial cell (HUVEC) library. The mature form of TweakR has only one hundred and two amino acids and six cysteine residues in its extracellular region. Five different assays demonstrate TWEAK-TweakR binding, and the interaction affinity constant (Kd) is within a physiologically relevant range of 2.3 +/- 0.1 nM. The TweakR cytoplasmic domain binds TRAFs 1, 2, and 3. Cross-linking of TweakR induces HUVEC growth, and mRNA levels are upregulated in vitro by a variety of agents and in vivo following arterial injury. Soluble TweakR inhibits endothelial cell migration in vitro and corneal angiogenesis in vivo.

Amino Acid Sequence↗

PubMed2000

The CXC chemokine receptor 2, CXCR2, is the putative receptor for ELR+ CXC chemokine-induced angiogenic activity.

We have previously shown that members of the ELR(+) CXC chemokine family, including IL-8; growth-related oncogenes alpha, beta, and gamma; granulocyte chemotactic protein 2; and epithelial neutrophil-activating protein-78, can mediate angiogenesis in the absence of preceding inflammation. To date, the receptor on endothelial cells responsible for chemotaxis and neovascularization mediated by these ELR(+) CXC chemokines has not been determined. Because all ELR(+) CXC chemokines bind to CXC chemokine receptor 2 (CXCR2), we hypothesized that CXCR2 is the putative receptor for ELR(+) CXC chemokine-mediated angiogenesis. To test this postulate, we first determined whether cultured human microvascular endothelial cells expressed CXCR2. CXCR2 was detected in human microvascular endothelial cells at the protein level by both Western blot analysis and immunohistochemistry using polyclonal Abs specific for human CXCR2. To determine whether CXCR2 played a functional role in angiogenesis, we determined whether this receptor was involved in endothelial cell chemotaxis. We found that microvascular endothelial cell chemotaxis in response to ELR(+) CXC chemokines was inhibited by anti-CXCR2 Abs. In addition, endothelial cell chemotaxis in response to ELR(+) CXC chemokines was sensitive to pertussis toxin, suggesting a role for G protein-linked receptor mechanisms in this biological response. The importance of CXCR2 in mediating ELR(+) CXC chemokine-induced angiogenesis in vivo was also demonstrated by the lack of angiogenic activity induced by ELR(+) CXC chemokines in the presence of neutralizing Abs to CXCR2 in the rat corneal micropocket assay, or in the corneas of CXCR2(-/-) mice. We thus conclude that CXCR2 is the receptor responsible for ELR(+) CXC chemokine-mediated angiogenesis.

Administration, Topical↗

PubMed2000

Nck-interacting Ste20 kinase couples Eph receptors to c-Jun N-terminal kinase and integrin activation.

The mammalian Ste20 kinase Nck-interacting kinase (NIK) specifically activates the c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase module. NIK also binds the SH3 domains of the SH2/SH3 adapter protein Nck. To determine whether Nck functions as an adapter to couple NIK to a receptor tyrosine kinase signaling pathway, we determined whether NIK is activated by Eph receptors (EphR). EphRs constitute the largest family of receptor tyrosine kinases (RTK), and members of this family play important roles in patterning of the nervous and vascular systems. In this report, we show that NIK kinase activity is specifically increased in cells stimulated by two EphRs, EphB1 and EphB2. EphB1 kinase activity and phosphorylation of a juxtamembrane tyrosine (Y594), conserved in all Eph receptors, are both critical for NIK activation by EphB1. Although pY594 in the EphB1R has previously been shown to bind the SH2 domain of Nck, we found that stimulation of EphB1 and EphB2 led predominantly to a complex between NIK/Nck, p62(dok), RasGAP, and an unidentified 145-kDa tyrosine-phosphorylated protein. Tyrosine-phosphorylated p62(dok) most probably binds directly to the SH2 domain of Nck and RasGAP and indirectly to NIK bound to the SH3 domain of Nck. We found that NIK activation is also critical for coupling EphB1R to biological responses that include the activation of integrins and JNK by EphB1. Taken together, these findings support a model in which the recruitment of the Ste20 kinase NIK to phosphotyrosine-containing proteins by Nck is an important proximal step in the signaling cascade downstream of EphRs.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Endothelial signal integration in vascular assembly.

Regulated assembly of a highly specialized interconnecting network of vascular endothelial and supportive cells is fundamental to embryonic development and organogenesis, as well as to postnatal tissue repair in metazoans. This review advances an "endotheliocentric" model that defines tasks required of endothelial cells and describes molecular controls that regulate steps in activation, assembly, and maturation of new vessels. In addition to the classical assembly mechanisms--angiogenesis and vasculogenesis--endothelial cells are also recruited into vascular structures from the circulatory system in adult animals and from resident mesenchymally derived progenitors during organogenesis of kidney and other organs. Paracrine signaling cascades regulated by hypoxia initiate a sequentially coordinated series of endothelial responses, including matrix degradation, migration, proliferation, and morphogenetic remodeling. Surface receptors on committed endothelial lineage progenitors transduce cues from extracellular-matrix-associated proteins and cell-cell contact to direct migration, matrix attachment, proliferation, targeting and cell-cell assembly, and vessel maturation. Through their capacity to spatially segregate and temporally integrate a diverse range of extracellular signals, endothelial cells determine their migratory paths, cellular partners, and life-or-death responses to local cues.

Animals↗

PubMed1999

Thromboxane A2 is a mediator of cyclooxygenase-2-dependent endothelial migration and angiogenesis.

Cyclooxygenase-2 (COX-2) inhibitors reduce angiogenic responses to a variety of stimuli, suggesting that products of COX-2 may mediate critical steps. Here, we show that thromboxane A2 (TXA2) is one of several eicosanoid products generated by activated human microvascular endothelial cells. Selective COX-2 antagonists inhibit TXA2 production, endothelial migration, and fibroblast growth factor-induced corneal angiogenesis. Endothelial migration and corneal angiogenesis are similarly inhibited by a TXA2 receptor antagonist, SQ29548. A TXA2 agonist, U46619, reconstitutes both migration and angiogenesis responses under COX-2-inhibited conditions. These findings identify TXA2 as a COX-2 product that functions as a critical intermediary of angiogenesis.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed1999

Surface densities of ephrin-B1 determine EphB1-coupled activation of cell attachment through alphavbeta3 and alpha5beta1 integrins.

Receptors of the Eph family and their ligands (ephrins) mediate developmental vascular assembly and direct axonal guidance. Migrating cell processes identify appropriate targets within migratory fields based on topographically displayed ephrin gradients. Here, EphB1 regulated cell attachment by discriminating the density at which ephrin-B1 was displayed on a reconstituted surface. EphB1-ephrin-B1 engagement did not promote cell attachment through mechanical tethering, but did activate integrin-mediated attachment. In endothelial cells, attachment to RGD peptides or fibrinogen was mediated through alphavbeta3 integrin. EphB1 transfection conferred ephrin-B1-responsive activation of alpha5beta1 integrin-mediated cell attachment in human embryonic kidney cells. Activation-competent but signaling-defective EphB1 point mutants failed to stimulate ephrin-B1 dependent attachment. These findings lead us to propose that EphB1 functions as a 'ligand density sensor' to signal integrin-mediated cell-matrix attachment.

Cell Adhesion↗

PubMed1999

Comparative analysis of different methodological approaches to the in vitro study of drug-induced apoptosis.

Apoptosis is a dynamic process in which a characteristic morphological or biochemical event used in an assay as a specific marker of apoptosis may be observed over a limited period of time. Asynchronous involvement of cells in apoptosis results in different proportions of apoptotic cells with blebbed membrane, broken nuclei, modified mitochondrial units or fragmented DNA coexisting in the culture at any single moment. Thus, depending on the method used, the extent of apoptosis determined in the same cell population may vary. In the present study, a microculture kinetic (MiCK) assay was used to monitor apoptosis in HL-60 cells exposed to 1, 2.5, 5, 10, and 20 micromol/L etoposide and cisplatin. Both the extent and timing of apoptotic responses were dependent on the drug and drug concentration. Time-lapse video microscopy (TLVM), flow cytometry analysis of the light scattering properties of cells, morphological studies of Giemsa-stained cells, annexin V binding, and DNA fragmentation assays were performed at multiple times of cell exposure to 10 micromol/L etoposide and 5 micromol/L cisplatin. Steep linear increases in optical density, indicating apoptosis in the MiCK assay, correlated with both linear increases in the proportion of cells with plasma membrane blebbing in TLVM and with increased side scattering properties of apoptotic cells in flow cytometry. During a 24-hour culture period, the MiCK assay and TLVM provided multiple consecutive appraisals of nondisturbed cell microcultures at intervals of 5 and 2.5 minutes, respectively, and thus could be considered as real time kinetic assays. With the three endpoint assays, each of which was applied 12 times at 2-hour intervals, maximum apoptotic responses varied from 22.5 to 72% in etoposide-treated cells and from 30 to 57% in cisplatin-treated cells. With the annexin V binding assay, maximum apoptosis could always be detected 4 to 5 hours earlier than it was seen in Giemsa-stained preparations and 8 hours earlier than it was detected by measuring of DNA fragmentation. Values of the maximum extent of apoptosis varied, being the lowest with annexin V and the greatest with DNA fragmentation assays. The best correlations of both extent and timing of apoptosis were observed between the MiCK, TLVM, and morphological assays. In conclusion, both a maximum apoptotic response and the time at which it was achieved are the obligatory requirements for determining the apoptosis-inducing potency of an agent and for comparing results of studies performed in different laboratories.

Annexin A5↗

PubMed1999

Endothelial localization of receptor tyrosine phosphatase, ECRTP/DEP-1, in developing and mature renal vasculature.

Developmental assembly of the renal microvasculature requires spatially and temporally coordinated migration, assembly, differentiation, and maturation of endothelial cells in the context of adjacent epithelial and mesangial cells. In this study, endothelial expression and distribution of the receptor tyrosine phosphatase ECRTP/DEP-1 were evaluated during and after developmental assembly of the renal microvasculature. Monoclonal antibodies against ECRTP/DEP-1 ectodomain epitopes localize its expression to membrane surfaces of endothelial cells in glomerular, peritubular capillary, and arterial renal sites of mature human and murine kidney. During kidney development, ECRTP/DEP-1 immunostaining is evident on a subpopulation of metanephric mesenchymal cells and on putative progenitors of glomerular capillary endothelial cells early in their recruitment to developing glomeruli. ECRTP/DEP-1 is prominently displayed on luminal membrane surfaces with punctate accumulations at inter-endothelial contacts that overlap with vascular endothelial-cadherin staining. ECRTP/DEP-1 is recruited to inter-endothelial contacts in confluent cultured human renal and dermal microvascular endothelial cells, yet experimental dissociation of vascular endothelial-cadherin from endothelial junctional complexes fails to redistribute ECRTP/DEP-1. These findings indicate that ECRTP/DEP-1 is expressed in anticipation of glomerular capillary endothelial recruitment during development, and suggest that ECRTP/DEP-1 ectodomain interacts with endothelial surface ligands that are engaged by cell-cell contact.

Animals↗

PubMed1998

Eph receptors discriminate specific ligand oligomers to determine alternative signaling complexes, attachment, and assembly responses.

Eph family receptor tyrosine kinases (including EphA3, EphB4) direct pathfinding of neurons within migratory fields of cells expressing gradients of their membrane-bound ligands. Others (EphB1 and EphA2) direct vascular network assembly, affecting endothelial migration, capillary morphogenesis, and angiogenesis. To explore how ephrins could provide positional labels for cell targeting, we tested whether endogenous endothelial and P19 cell EphB1 (ELK) and EphB2 (Nuk) receptors discriminate between different oligomeric forms of an ephrin-B1/Fc fusion ligand. Receptor tyrosine phosphorylation was stimulated by both dimeric and clustered multimeric ephrin-B1, yet only ephrin-B1 multimers (tetramers) promoted endothelial capillary-like assembly, cell attachment, and the recruitment of low-molecular-weight phosphotyrosine phosphatase (LMW-PTP) to receptor complexes. Cell-cell contact among cells expressing both EphB1 and ephrin-B1 was required for EphB1 activation and recruitment of LMW-PTP to EphB1 complexes. The EphB1-binding site for LMW-PTP was mapped and shown to be required for tetrameric ephrin-B1 to recruit LMW-PTP and to promote attachment. Thus, distinct EphB1-signaling complexes are assembled and different cellular attachment responses are determined by a receptor switch mechanism responsive to distinct ephrin-B1 oligomers.

Animals↗

PubMed1998

Nck recruitment to Eph receptor, EphB1/ELK, couples ligand activation to c-Jun kinase.

Eph family receptor tyrosine kinases signal axonal guidance, neuronal bundling, and angiogenesis; yet the signaling systems that couple these receptors to targeting and cell-cell assembly responses are incompletely defined. Functional links to regulators of cytoskeletal structure are anticipated based on receptor mediated cell-cell aggregation and migratory responses. We used two-hybrid interaction cloning to identify EphB1-interactive proteins. Six independent cDNAs encoding the SH2 domain of the adapter protein, Nck, were recovered in a screen of a murine embryonic library. We mapped the EphB1 subdomain that binds Nck and its Drosophila homologue, DOCK, to the juxtamembrane region. Within this subdomain, Tyr594 was required for Nck binding. In P19 embryonal carcinoma cells, activation of EphB1 (ELK) by its ligand, ephrin-B1/Fc, recruited Nck to native receptor complexes and activated c-Jun kinase (JNK/SAPK). Transient overexpression of mutant EphB1 receptors (Y594F) blocked Nck recruitment to EphB1, attenuated downstream JNK activation, and blocked cell attachment responses. These findings identify Nck as an important intermediary linking EphB1 signaling to JNK.

Adaptor Proteins, Signal Transducing↗

PubMed1998

Origins and formation of microvasculature in the developing kidney.

Regulation of microvessel assembly in the developing kidney is not known and may occur through vasculogenic, angiogenic, or both processes. To examine this question, we grafted rat and mice embryonic (E) day 12 (E12) kidneys, which have only a rudimentary vasculature, into anterior eye chambers of mouse and rat hosts. Species-specific, monoclonal anti-basement membrane antibodies showed that glomerular basement membranes, mesangial matrices, and microvessel basement membranes were always derived from the graft. When wild-type E12 mouse kidneys were grafted into anterior chambers of ROSA26 mice, in which the beta-galactosidase transgene is expressed ubiquitously, glomerular and microvascular endothelial cells stemmed from the graft, even after maintenance of kidneys in organ culture for 6 days before grafting. Immunolabeling with antibodies against the vascular endothelial growth factor (VEGF) receptor, Flk1, the EphB1 receptor, and its ligand, ephrin-B1, labeled discrete mesenchymal cells in embryonic and newborn kidney cortex, as well as developing microvessel and glomerular endothelium. In adult kidneys, Flk1 labeled glomeruli weakly, other vascular structures were unlabeled. When wild-type E12 kidneys were grafted under renal capsules of adult ROSA26 hosts, endothelium developing within the graft again came from the implanted kidney. In contrast, when E12 kidneys were grafted into renal cortices of newborns, glomeruli within grafts now contained host-derived endothelium. Similarly, when ROSA26 E12 kidneys were implanted into newborn wild-type hosts, chimeric vessels containing graft- and host-derived endothelium were seen in nearby host tissue. Our results indicate that cells capable of forming the entire microvascular tree of grafted metanephroi are already present in the E12 kidney. We hypothesize that Flk1/VEGF and EphB1/ephrin-B1 mediate renal endothelial mitosis-motility and cell guidance-aggregation behavior, respectively.

Animals↗

PubMed1998

Renal microvascular assembly and repair: power and promise of molecular definition.

Developmental assembly of the renal microcirculation is a precise and coordinated process now accessible to experimental scrutiny. Although definition of the cellular and molecular determinants is incomplete, recent findings have reframed concepts and questions about the origins of vascular cells in the glomerulus and the molecules that direct cell recruitment, specialization and morphogenesis. New findings illustrate principles that may be applied to defining critical steps in microvascular repair following glomerular injury. Developmental assembly of endothelial, mesangial and epithelial cells into glomerular capillaries requires that a coordinated, temporally defined series of steps occur in an anatomically ordered sequence. Recent evidence shows that both vasculogenic and angiogenic processes participate. Local signals direct cell migration, proliferation, differentiation, cell-cell recognition, formation of intercellular connections, and morphogenesis. Growth factor receptor tyrosine kinases on vascular cells are important mediators of many of these events. Cultured cell systems have suggested that basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF) promote endothelial cell proliferation, migration or morphogenesis, while genetic deletion experiments have defined an important role for PDGF beta receptors and platelet-derived growth factor (PDGF) B in glomerular development. Receptor tyrosine kinases that convey non-proliferative signals also contribute in kidney and other sites. The EphB1 receptor, one of a diverse class of Eph receptors implicated in neural cell targeting, directs renal endothelial migration, cell-cell recognition and assembly, and is expressed with its ligand in developing glomeruli. Endothelial TIE2 receptors bind angiopoietins (1 and 2), the products of adjacent supportive cells, to signals direct capillary maturation in a sequence that defines cooperative roles for cells of different lineages. Ultimately, definition of the cellular steps and molecular sequence that direct microvascular cell assembly promises to identify therapeutic targets for repair and adaptive remodeling of injured glomeruli.

Animals↗

PubMed1997

Identification of a subpopulation of human renal microvascular endothelial cells with capacity to form capillary-like cord and tube structures.

Endothelial specialization is a prominent feature within distinct capillary beds of organs such as mammalian kidney, yet immunological markers for functionally distinct subpopulations of cultured endothelial cells from tissue sources such as kidney have not been available. We developed a simple and reproducible isolation and culture procedure to recover human renal microvascular endothelial cells (HRMEC) from the cortex of unused donor kidneys. This procedure yields highly purified preparations of cells that display endothelial markers that include Factor VIII antigen, acetyl-LDL receptors, and determinants that bind Ulex europaeus lectin. HRMEC assemble into capillary-like cord and tube structures when plated on the surface of basement membrane-like matrix (BMM) in media containing phorbol myristate acetate. To further define subpopulations of HRMEC, we generated a panel of monoclonal antibodies and screened for those recognizing cell surface determinants. One monoclonal antibody recovery from this screen recognized a cell surface protein expressed on a subpopulation of HRMEC that we have designated PEC-1 (pioneer endothelial cell antigen-1). Cells expressing PEC-1 extended long, interconnecting filopodial processes in response to phorbol myristate acetate and assembled into capillary-like structures when plated on BMM. Anti-PEC-1 immunoprecipitated proteins of 25 and 27 kDa. Magnetic bead separation of PEC-1 (+) cells selected cells that assemble into capillary-like cord and tube structures. The remaining PEC-1 (-) HRMEC population formed matrix adherent patches. In the kidney, the PEC-1 determinant is expressed on a small subpopulation of microvascular glomerular cells and is prominently expressed on the apical membrane of proximal tubule cells. The PEC-1 determinant discriminates among subpopulations of HRMEC, identifying a subpopulation that contributes to assembly of capillary-like structures.

Adult↗

PubMed1996

Ligand activation of ELK receptor tyrosine kinase promotes its association with Grb10 and Grb2 in vascular endothelial cells.

ELK is a member of the Eph-related tyrosine kinase family that includes receptors signaling axonal guidance, neuronal bundling, and angiogenesis. We recently identified ELK expression in human renal microvascular endothelial cells and sought to identify intracellular proteins through which it signals responses. The cytoplasmic domain of ELK was used as "bait" in a yeast two-hybrid screen to identify interactive proteins expressed from a randomly primed embryonic murine library (E9.5-10.5). Among interactive products of 76 cDNAs characterized, 10 nonidentical, overlapping clones encoded the SH2 domain of the recently reported Grb10 adapter protein, and an additional 3 encoded Grb2. A self-phosphorylated recombinant, baculovirus-expressed GST-ELKcy fusion protein bound Grb10 and Grb2 from human renal microvascular endothelial cell extracts, while the unphosphorylated fusion form did not. Site-directed mutation identified Tyr-929 as a putative phosphorylation site required for Grb10, but not Grb2, interaction in yeast and recombinant protein assays. The ELK ligand, LERK-2/Fc, stimulated tyrosine phosphorylation of ELK, and recruitment of Grb10 and Grb2 to endothelial ELK receptors recovered by wheat germ agglutinin lectin and immunoprecipitation. These findings define ligand-activated interaction between ELK and the SH2 domains of Grb2 and the newly identified Grb10 protein that shares homology with a Caenorhabditis elegans gene product implicated in neural cell migration.

Adaptor Proteins, Signal Transducing↗

PubMed1996

Detergent solubility defines an alternative itinerary for a subpopulation of PDGF beta receptors.

Current models of platelet-derived growth factor (PDGF) beta receptor itinerary are based upon the properties of receptors recovered from nonionic detergent-solubilized cellular extracts. Comparing several commonly used cell extraction procedures, we have determined that up to 50% of immunoreactive PDGF beta receptors, reside in a Triton X-100 insoluble pool in a wide distribution of cultured cell lines, including Balb/c-3T3, NIH 3T3, and Swiss fibroblasts, primary murine and human fibroblasts, and primary human glial cells. Many properties of Triton insoluble receptors are distinct from the well-characterized PDGF beta receptors, including 1) delayed arrival of newly synthesized receptors into the Triton insoluble fraction, 2) prolonged half-life in the presence of PDGF, 3) increased abundance with increasing cell density, 4) inaccessibility to modification by extracellular compartment enzymes, 5) cofractionation with cytoskeletal proteins, and 6) a higher basal tyrosine phosphorylation state. PDGF stimulates accumulation of tyrosine phosphorylated PDGF beta receptors in the Triton X-100 insoluble fraction. Cell surface PDGF beta receptors modified by enzymatic desialylation redistribute to the insoluble fraction. These findings distinguish the itinerary of a large subpopulation of PDGF beta receptors from those characterized previously. Receptors in this fraction represent a long-lived tyrosine phosphorylated population that may effect responses for extended periods following ligand activation.

Animals↗

PubMed1996

ELK and LERK-2 in developing kidney and microvascular endothelial assembly.

Eph family receptor tyrosine kinases direct neuronal cell targeting, bundling and intercellular aggregation activity, yet their role in mammalian kidney development has been unexplored to date. We recently identified expression of ELK (Eph-like kinase) receptors in cultured human renal microvascular endothelial cells (HRMEC), and showed that ELK mediates their in vitro assembly into capillary-like structures in response to the exogenous ligand, LERK-2. Here we identify expression of the ELK ligand, LERK-2, in HRMEC and in primitive vascular structures of developing murine kidney. ELK and LERK-2 are expressed on endothelial progenitor cells of primitive microvasculature in a pattern similar to that of the VEGF receptor, flk-1. ELK LERK-2 and flk-1 antigens are also displayed on the branching ureteric bud epithelium; ELK and LERK-2 expression persists in mature collecting ducts, glomeruli and arterioles. To explore whether renal-derived endothelial cells may distinguish LERK-2 from the angiogenic Eck ligand, LERK-1 (B61), and whether endothelial cells from different sources may distinguish among Eph receptor ligands, we compared HRMEC and human umbilical vein endothelial cell (HUVEC) responses in an in vitro capillary-like assembly assay. HRMEC endothelial cells assembled capillary-like structures in response to LERK-2, but not LERK-1, under conditions that promoted HUVEC to assemble in response to LERK-1, but not LERK-2. Therefore, responses mediated through specific Eph family receptors (ELK and Eck) are discriminated by endothelial cells from different vascular bed sources. ELK and its ligand, LERK-2, are spatially and temporally coordinated in expression and may function in morphogenesis of the renal microvasculature.

Animals↗

PubMed1993

Endothelial heterogeneity in Shiga toxin receptors and responses.

This study addresses the basis for regional microvascular susceptibility to bacterial toxins implicated in hemolytic uremic syndrome. The results indicate a relationship between the degree of Shiga toxin sensitivity of human endothelial cells from different sources and the amount of globotriaosylceramide (Gb3) glycosphingolipid receptor for Shiga toxin expressed by these cells. Cell viability and protein synthesis of renal endothelial cells were reduced to 50% by 1 pM Shiga toxin, while umbilical vein cells were not affected by > 1 nM toxin. Similarly, basal levels of Gb3 were approximately 50 times higher in renal endothelial cells than in the umbilical endothelial cells. Pre-exposure of umbilical endothelial cells to tumor necrosis factor-alpha or bacterial lipopolysaccharide increased Gb3 content 4-6-fold coincident with increases in sensitivity to cytotoxic and protein synthesis inhibitory effects of Shiga toxin. Lipopolysaccharide induction of both Gb3 and sensitivity to Shiga toxin cytotoxic action in umbilical endothelial cells was dependent on the structure of lipopolysaccharide. Neither tumor necrosis factor-alpha nor lipopolysaccharide altered the Shiga toxin sensitivity or the Gb3 content of renal endothelial cells. These data indicate that differential endothelial expression of glycolipid receptors for Shiga toxins may be responsible for localized involvement of the kidney in hemolytic uremic syndrome.

Bacterial Toxins↗

PubMed1993

A western blot assay detects autoantibodies to cryptic endothelial antigens in thrombotic microangiopathies.

Autoantibodies detected by immunofluorescence, ELISA, and complement-fixation techniques have provided discriminatory markers for many human diseases. However, these commonly applied assays may fail to detect antibodies against antigenic sites which are either inaccessible or not displayed in recognizable cellular structures. Moreover, molecular identities of recognized antigen(s) are not determined with such methods. We have used Western blot analysis of cellular proteins derived from human renal microvascular endothelial cells (HRMEC) to identify autoantibodies in patients with pathological endothelial injury. Exploring the possibility that endothelial injury may expose cryptic endothelial antigens to immune recognition, we detected antibodies binding a number of distinct HRMEC proteins. Among these, antibodies recognizing specific HRMEC proteins of 43 kDa were commonly detected in plasmas from patients with thrombotic thrombocytopenic purpura (TTP) (13 of 14) and hemolytic uremic syndrome (HUS) (4 of 5) but were absent in 9 of 10 healthy subjects and 11 patients with a range of diseases not associated with endothelial injury or insult. Antibodies binding 43-kDa HRMEC antigens were detected in individual patients with systemic lupus erythematosus, anti-glomerular basement membrane nephropathy, and heparin-associated thrombocytopenia, as well as in one of three patients with immune thrombocytopenic purpura. Similar antibodies were detected in one hypercholesterolemic subject. Antibodies from four TTP patients were affinity purified and shown by two-dimensional analysis to recognize 43-kDa proteins having identical pl's (5.9, 6.0, and 6.1). Subcellular fractionation localized these antigens to cytosolic and nuclear compartments, sites presumably protected from immune recognition in the absence of endothelial injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoantibodies↗